1. Field
This invention relates generally to microbial growth media and specifically to an improved isolation medium for the rapid detection of pathogenic Neisseria, Neisseria gonorrhoeae and Neisseria meningitidis, the causative agents of gonnorrhea and cerebrospinal meningitis diseases.
2. Prior Art
Diagnosis of infection by pathogenic Neisseria requires the use of a suitable culture medium, one which will permit the in vitro growth of the pathogenic Neisseria and inhibit the growth of other bacteria. Typically, in the prior art, a specimen is inoculated onto the surface of a suitable medium and incubated under suitable conditions for a period of time up to 48 hours. Pathogenic Neisseria which are present in the specimen will grow and form colonies which can then be detected by visual inspection.
Various types of culture media have been utilized in the prior art. For example, Simpson et al, U.S. Pat. No. 4,039,387, relates to a growth medium for distinguishing between Neisseria gonorrhoeae and Neisseria meningitidis.
The particular medium most widely used in the art at the time of this application is known as the Thayer-Martin medium. This medium was disclosed in 1966 by Messrs. Thayer and Martin in Public Health Rep. 81: 559-562. It has been observed that the Thayer-Martin medium is less than 100 percent effective in isolating N. gonorrhoeae, isolation rates ranging from about 40 percent to about 90 percent. This means that only about 40 to 90 percent of the specimens known to contain the bacteria will show growth. In a clinical situation, this means that from 10 percent to 50 percent of all individuals infected with the disease will remain undetected after observation of the first culture.
In view of the increasing incidence of the disease to epidemic proportions and further in view of the very large numbers of cases, both reported and unreported, the difference in detection rates is very important. It is perhaps even more important with respect to the other pathogenic Neisseria, N. meningitidis, since this bacteria causes a potentially fatal disease the prompt and accurate detection of which is, in many cases, critical.
It is known in the prior art to use cations such as ferric ions or aluminum ions in a medium for the detection of pathogenic Neisseria in order to increase the growth of the bacteria. For example, in U.S. Pat. No. 3,936,355, Lawson discloses the use of ferric nitrate in a culture medium for supporting the growth of certain types of microorganisms, including Neisseria gonorrhoeae and Neisseria meningitidis. The standard practice in the prior art has been to add iron to the culture medium in the form of ferric nitrate.
Kellogg et al investigated Neisseria gonorrhoeae and in their paper, Neisseria Gonorrhoeae, J. Bacteriology, September 1968, 596, identified four distinct colonial forms of gonococcus, T1, T2, T3 and T4. The former two types were shown to be virulent to man and the latter two to be relatively avirulent. At page 600, Kellogg et al observed the importance of including ferric ions in the culture medium to promote growth of the bacteria. Kellogg et al point out that the types of anions accompanying the ferric ions had no effect on the results, and that while glucose alone was ineffective to promote increased growth, use of glucose with ferric ions produced "an additive effect". In FIG. 4, at Page 600 of their paper, Kellogg et al plotted the growth stimulation in colony diameter with the inclusion of ferric ions, with the inclusion of glucose, and with the inclusion of a mixture of glucose and ferric ions wherein the ratio of glucose to ferric ions was 1.0. These results indicate that the increase in colony diameter in the medium is only slightly greater when a glucose/ferric combination is utilized compared to the use of ferric ions alone. Further, Kellogg et al point out at Page 600 of their paper that "a ratio of glucose to ferric ions of less than one depressed the colony size to a level intermediate between that obtained with either additive alone."
In their paper entitled "Pathogenesis and Immunology of Experimental Gonococcal Infection: Role of Iron in Virulence," (1975, Infect. Immun. 12: 1313-1318), applicants disclosed the creation of an experimental animal model, chicken embryos, with findings that the colonial types previously identified as virulent (T1 and T2) killed the animals, and that the forms identified as relatively nonvirulent (T3 and T4) did not. Applicants further disclosed in that paper that the virulence of the T3 and T4 types to chicken embryos is enhanced by adding iron in various forms, including in the form of an iron-dextran complex (specifically, Imferon).
The present invention of applicants was first disclosed in their paper entitled "Imferon Agar: Improved Medium for Isolation of Pathogenic Neisseria," J. Clinical Microbiology, Volume 6, Number 3, September 1977, pp. 293-297.
It has been estimated that there are some three to four million cases of gonorrhea each year in the United States, most of which go unreported. Improvement in the rate of detection of gonorrhea from a first culture would represent a significant advancement in the art. Such improvement can be obtained by devising a method and composition for effectively increasing the colony size of the bacteria Neisseria gonorrhoeae so that the presence of the bacteria may be readily observed within a short period of time. Similar advantages would also result if an improved medium could be developed for the detection of the other pathogenic Neisseria, Neisseria meningitidis.
This invention seeks to develop such methods and such a medium to improve the speed and rate of isolation of Neisseria gonorrhoeae and Neisseria Meningitidis, so that their colonies are much larger in size and will appear after a much shorter incubation period.